The present invention pertains to a method for histological staining of arteriovenous malformation (AVM) tissue samples in order to quantify parameters of surgical AVM embolization using an europium aryl-xcex2-diketone complex.
Arteriovenous malformations (AVMs) are, in most patients, congenital lesions formed by tangled networks of blood vessels. The cause of AVMs is not known, but most AVMs are thought to be due to abnormal development of blood vessels during fetal development. While AVMs can potentially form anywhere in the body, those formed in the brain are particularly problematic.
In normal brain tissue, blood enters through major cerebral arteries, passes through smaller arterioles, and subsequently moves into capillaries. Capillaries are tiny blood vessels that allow blood to deliver necessary oxygen and nutrients to the brain and remove waste products of brain metabolism. Normally, after passing through the capillaries, the blood enters the brain""s venous system. When an AVM exists in the brain, blood is shunted directly from the arterial system to the venous system. There is normally a drop in pressure as blood travels from arteries to veins, but when an AVM is present, the rate of blood flow from arteries to veins can be high and the pressure can thus be elevated within the veins. This elevated pressure can contribute to a variety of complications, including stroke, seizures, bleeding, and disruption of the normal function of brain cells near the AVM.
The treatment for AVMs includes a procedure by which an endovascular occlusion of blood flow through these abnormal arteriovenous shunts is intentionally created by physician intervention. This is done by the surgical introduction of an occlusive agent into the AVM to embolize the AVM such that the blood flow through the AVM is impeded and ultimately rerouted to the venous system.
Over the past several years, many different occlusive agents have been tested in a variety of endovascular treatments. Liquid adhesive polymers (glues) which rapidly solidify have been used for such procedures as bone grafting, skin incision repair, dental cements, and as embolic agents. More specifically, various derivatives of alkyl-cyanoacrylates have been demonstrated to provide highly favorable results when compared to other embolic agents.
The most common embolic agent used currently is n-butyl 2-cyanoacrylate (NBCA) or enbucrilate. In order to control the polymerization time of NBCA, as well as to opacify the mixture for angiographic visualization, an iodized poppyseed oil-based contrast agent (Lipiodol) is added to the NBCA. Glacial acetic acid (GAA) is also used to delay the polymerization time further by decreasing the pH of the mixture.
Research efforts have focused on finding ways to better quantify the hemodynamic parameters, such as pressure, flow and NBCA injection pressure involved in AVM embolization, thus improving the reproducibility of successful outcomes for patients. An understanding of the complex and variable anatomy of AVMs is essential prior to treatment, but knowledge of the velocities and transit times of blood through the AVM nidus (arteriovenous fistulae network) is equally as essential for successful endovascular intervention. Both physical skill as well as precise prediction of embolic agent behavior within the vessels is needed in order to avoid non-optimal glue casting outcomes.
During injection of the NBCA, polymerization of the NBCA-Lipiodol mixture exactly within the AVM nidus is crucial. If proximal feeding arteries are occluded without NBCA penetration into the nidus, revascularization of the AVM will occur. Delayed polymerization and occlusion of distal draining veins may result in serious complications such as hemorrhage, emboli, stroke or death.
Since the hemodynamic parameters involved have not been accurately quantified to date, NBCA embolization of AVMs is still considered a high-risk procedure. Improved characterization of embolization hemodynamics necessitates analysis of how deep NBCA penetrates into the AVM nidus. In addition, interaction of the mixture (NBCA and Lipiodol) with the vessel wall and polymerization rates must be examined. Thus, there is a need for better visualization techniques of embolized AVM tissue samples so that such parameters can be quantified.
The present invention comprises a new histological staining technique that allows quantification of previously unmeasured parameters involved in surgical AVM embolization. The invention allows the evaluation of the polymerization characteristics of various ratios of embolization agents, such as Lipiodol/NBCA/glacial acetic acid (GAA) mixtures, by virtue of a new tissue sample preparation protocol and staining technique.
To determine the depth of NBCA penetration within the AVM model and to characterize the polymerization patterns of various mixtures within a model vessel, histologic cross- and longitudinal sections were prepared for microscopy using a new staining method comprising the use of a europium aryl-xcex2-diketone complex and petroleum ether. Paraffin-embedded tissue sections were subjected to the staining protocol to improve differentiation between NBCA and Lipiodol. Quantification of NBCA and Lipiodol within the lumen of rete cross-sections was accomplished using image analysis software to determine percent luminal area occluded by embolization. Upon application of europium tris(thenoyltrifluoroacetone) or TEC, intense europium fluorescence was seen when the tissue samples were excited by low-power ultraviolet light. The area of europium intensity within the lumen corresponded to NBCA concentration, and addition of GAA aided the NBCA distribution throughout the lumen without affecting fluorescence intensity. The invention demonstrates that NBCA can be easily differentiated from Lipiodol and quantification of previously unmeasured parameters can be readily performed on these sections because of the improved tissue sample staining technique.
The invention also relates to a kit for quantifying n-butyl 2-cyanoacrylate (NBCA) within treated tissues that includes europium aryl-xcex2-diketone complex, petroleum ether and methanol, each in containers holding predetermined amounts of the reagents.